Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry...
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Transcript of Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry...
![Page 1: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.](https://reader035.fdocuments.net/reader035/viewer/2022062320/56649d545503460f94a30da7/html5/thumbnails/1.jpg)
Analysis and Sorting of T reg Analysis and Sorting of T reg Cells on the FACSAriaCells on the FACSAria
John DaleyJohn DaleyDirector of Flow CytometryDirector of Flow Cytometry
Hematologic NeoplasiaHematologic NeoplasiaDana-Farber Cancer InstituteDana-Farber Cancer Institute
Harvard Medical SchoolHarvard Medical School
http://www.bdbiosciences.com/features/products
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http://www.bdbiosciences.com/features/products/display_product.php?keyID=162
From BD Biosciences WebsiteFeatured products:Regulatory T cells
![Page 3: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.](https://reader035.fdocuments.net/reader035/viewer/2022062320/56649d545503460f94a30da7/html5/thumbnails/3.jpg)
The way we used to sort Human The way we used to sort Human CD4+CD25+ PBL’s on the CD4+CD25+ PBL’s on the
FACSAriaFACSAria
SORT TOP2- 3% CD25 HI
![Page 4: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.](https://reader035.fdocuments.net/reader035/viewer/2022062320/56649d545503460f94a30da7/html5/thumbnails/4.jpg)
Then CD45Ra was added to the Then CD45Ra was added to the Sort DesignSort Design
![Page 5: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.](https://reader035.fdocuments.net/reader035/viewer/2022062320/56649d545503460f94a30da7/html5/thumbnails/5.jpg)
Four way reg sort
Multicolor(5) Minor populations : Four way sorting: Treg Story
CD45ra FITCCD127 PECD25 PE-CY5CD4 PE-CY7CD3 PAC BLUE
![Page 6: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.](https://reader035.fdocuments.net/reader035/viewer/2022062320/56649d545503460f94a30da7/html5/thumbnails/6.jpg)
Compensation ComparisonsCompensation Comparisons
Manual Comp with cells and actual Manual Comp with cells and actual reagentsreagents
Autocomp with cells and actual reagentsAutocomp with cells and actual reagents
Autocomp with beads and actual reagentsAutocomp with beads and actual reagents
Autocomp with beads and CD45+ Autocomp with beads and CD45+ reagentsreagents
Autocomp with cells and CD45+ reagentsAutocomp with cells and CD45+ reagents
![Page 7: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.](https://reader035.fdocuments.net/reader035/viewer/2022062320/56649d545503460f94a30da7/html5/thumbnails/7.jpg)
BD cells Cell Autocomp
BD cellsBead AutoComp
![Page 8: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.](https://reader035.fdocuments.net/reader035/viewer/2022062320/56649d545503460f94a30da7/html5/thumbnails/8.jpg)
Treg Post Sort Reanalysis
![Page 9: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.](https://reader035.fdocuments.net/reader035/viewer/2022062320/56649d545503460f94a30da7/html5/thumbnails/9.jpg)
99.4 97.198.4 99.9
POST SORT REANALYSIS: HUMAN PBL CD3+CD4+ CD45RA+ &CD45RA- Treg+/Teffector+/ --
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Actual reagents cells compensation
cells
beads
Actual reagents beads compensation
cells beads
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FoxP3 Expression in CD4+ Treg FoxP3 Expression in CD4+ Treg CD45RA+CD45RA+
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Fluorescence minus 1Fluorescence minus 1